Abstract

Abstract The activity of the enzyme adenylate cyclase has been determined in normal rat kidney (NRK) cells and NRK cells transformed by either the Kirsten sarcoma virus (Ki-SV) or the Moloney sarcoma virus (Mo-SV). The specific activity of the NRK enzyme increases with increasing cell population density. Transformation apparently decreases this response, for the cyclase activity from KNRK cells (NRK cells transformed by Ki-SV) is low and rises only slightly with increasing cell density. The activity in transformed cells is at most 50% that noted in confluent NRK cells. Further, when assayed in the presence of 10 mm NaF, the KNRK and MNRK (NRK cells transformed by Mo-SV) cyclase activity is appreciably lower than that from NRK cells. These changes in adenylate cyclase activity are not due to an altered affinity for ATP; the enzymes from NRK, KNRK, and MNRK cells all exhibit an apparent Km (ATP) ∼0.24 mm. The activity of the NRK enzyme increases with increasing Mg2+ concentration through 15 mm; the enzyme from KNRK and MNRK cells is saturated at 1 mm Mg2+. When assayed in the presence of Mn2+, the cyclase activity of KNRK and MNRK cells is decreased relative to NRK cells. The effects of GTP and prostaglandin E1 were also studied. The adenylate cyclase of NRK homogenates is stimulated 2- to 3-fold by prostaglandin E1; GTP has little effect (∼20% stimulation) on either basal or protaglandin E1-stimulated activity. However, the activity of crude, washed NRK membranes is enhanced 2- to 3-fold by GTP at 10-6 to 10-4 m. Stimulation by prostaglandin E1 is dependent upon the presence of GTP in this membrane preparation. Of particular interest is the observation that the cyclase from KNRK and MNRK cells, either homogenates or membrane preparations, is unresponsive to prostaglandin E1 in either the presence or absence of GTP. Our studies indicate that transformation of fibroblasts with RNA tumor viruses often results in decreased adenylate cyclase activity; the mechanism by which this reduction in enzyme activity is accomplished appears to differ with different viruses.

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